Tumor Necrosis Factor Alpha -308 G/A Single Nucleotide Polymorphism and Risk of Sperm Abnormalities in Iranian Males

Background Signaling molecules such as cytokines regulate spermatogenesis during the maturation of germ cells and sperm apoptosis. Tumor necrosis factor alpha (TNFα) is one of the most-documented cytokines that is involved in spermatogenesis. We investigated the association of the TNFα -308 G/A single nucleotide polymorphism with sperm abnormalities in Iranian males. Materials and Methods This case-control study included 180 infertile men who re- ferred to Yazd Research and Clinical Center for Infertility and 100 healthy normospermic controls. Infertile men were classified into four groups of azoospermia (n=91), oligospermia (n=26), teratospermia (n=30) and asthenoteratospermia (n=33). After sperm analysis, DNA was extracted from blood and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out for the genotyping of TNFα- 308 G/A. Results The A allele was significantly associated with sperm abnormality in our population [(P<0.001, odds ratios (OR) 95% confidence interval (CI)=2.31]. In addition, the A allele was also associated with azoospermia (P<0.001, OR (95% CI)=2.484), oligospermia (P=0.005, OR (95% CI)=2.51) and teratospemia (P<0.001, OR (95% CI)=3.385) but not with asthenoteratospermia (P=0.623). Conclusion Our data suggest that this single nucleotide polymorphism (SNP) maybe associated with the risk of sperm abnormality in infertile men of Iranian origin.


Introduction
It is estimated that about 15% of couples globally suffer from infertility. Male infertility constiare mainly responsible (1, 2). Other causes of male infertility maybe related to post-testicular obstruction, endocrine dysfunction and vascular abnormalities (3). During the last decade, it has become clear that some signaling molecules that mediate the intercellular communication and integration have an important role in the hormonal regulation of germ cell maturation in testis. Cytokines are mediator molecules that are involved in this regulation and as a result have an important impact on spermatogenesis (4, 5). Among cytokines, most studied molecule but also the most potent in germ cell apoptosis, peritubular cell secretion and regulation of spermatogenesis (6). Its receptors are present in Sertoli and Leydig cells, allowing Some studies have shown a negative association terone production, which has a direct impact on male infertility, has also been reported in some ex- The gene as a single copy gene is located on chromosome 6p21.3 within the major histocompatibility complex (MHC) gene cluster (12). Gene variation such as single nucleotide polymorphisms (SNPs) in C/A, -857 C/T, -575 G/A, -376 G/A, -244 G/A and -238 G/A in the promoter region of the gene have promoter region of has been implicated to increase promoter activity, leading to an increased with sperm motility and morphology (9,16,17). Zalata et al. (18) showed the association of the and decreased sperm motility, count, morphology, acrosin activity and seminal a-glucosidase. Shukla et al. (19) showed that there is a strong association between this SNP and male infertility in the Indian populations of Uttar Pradesh.
regulator of steroidogenesis and may affect spermatogenesis, we investigated the association of the abnormality in infertile males of Iranian origin.
mospermic individuals as the control group. The case individuals were recruited from Yazd Research and Clinical Center for Infertility from Sep-ed based on sperm abnormality into azoospermia approved by the Ethics Committee of Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Written informed consent was obtained from each individual. All semen analysis and clinical examinations were done according to the World Health Genomic DNA was extracted from whole blood samples using the salting out method. We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) F: 5'-AGGCAATAGGTTTTGAGGGCCAT-3' and R: 5'-TCCTCCCTGCTCCGATTCCG-3' prim-

Results
In this study, PCR-RFLP was able to identify both region of gene (Fig.1).  Table 1 shows the related parameters of each group. The frequencies of alleles and genotypes and their association with the studied group are listed in Table 2. Ancestral genotype GG and allele G were taken as reference. Statistical analybetween this SNP and the AZ, O and T patient groups but not with the AT group. The frequency of the AA genotype was 13% in the healthy normospermic (N) group, 27.4% in the AZ group 24.2% of the AT group.

Discussion
Genetic variation such as SNPs in promoter region may affect its expression. There are several studies that have investigated the association of SNPs with different diseases such as colorectal cancer, pre-eclampsia, prostate cancer and Crohn's disease (21,22). The basic knowl-genesis is based on the study by Suh et al. (23) in spermatogenesis, reduced testis weight and sperm count in comparison with wild-type mice.
SNP to identify its possible association with sperm abnormality in Iranian males. To the best of our knowledge, this study has not been undertaken in gospermia and teratospermia. In other words, this SNP is among many genetic factors that may lead to a decreased count of sperm and abnormal morphology in our cases.
Similar to our study, Tronchon et al. (13) also allele with oligospermia and teratospermia. Zalata et al. (18) also observed an increased frequency of pared with the infertile group in the Egyptian population. In the Indian population, consistently, the frequency of the AA genotype was higher in infertile individuals rather than fertile subjects, and higher level of apoptosis and necrosis levels were observed in infertile males, likely due to increased levels of reactive oxygen species (19). In contrast, C>T and -863 C>A SNPs with sperm abnormalities (asthenozoospermia and oligo-asthenoteratozoospermia) in the Australian population. In the Greek population, Lazaros et al. (25) also found no association between -863 C>A and semen quality. The differences between the results of studies can be related to different number of studied individuals and different studied population with subgroups and ethnicities. known to affect spermatogenesis by changing the structure of the blood-testis barrier and apical ectoplasmic specialization of Sertoli cells, which may lead to abnormal spermatogenesis (26). Moreo-ver, it affects the Fas ligand system and germ cell apoptosis which have important roles in the germ cell maturation and normal spermatogenesis (27, receptor activates signaling molecules in the transduction pathway, in which adaptor proteins interact with conserved death domains. The adaptor proteins increase activation of caspase-8 causing the release of cytochrome c from mitochondria. This lar weight complex (apoptotic protease activating factor-1, cytochrome C, and caspase-9) that actistudy certainly has its own limitations and further association studies with more polymorphisms and individuals may provide more representative data on the association of variation with different sperm abnormalities in the Iranian population.

Conclusion
Our study shows that there is a positive associasperm abnormalities in the Iranian population. Given that the A allele leads to increased exprestreatment for male infertility.